The present invention relates to new nucleptide sequences coding for variable regions of xcex1 chain T-cell receptors, corresponding peptide segments and the diagnostic and therapeutic uses.
It is known that the receptors recognizing antigens at the surface of mature T lymphocytes (hereafter designated T-cell receptors) possess a structure having a certain similarity with those of immunoglobulins. Therefore, they contain heterodimeric structures containing xcex1 and xcex2 glycoprotein chains or xcex3 and xcex4 glycoprotein chains (see Meuer et al. (1), Moingeon et al. (2), Brenner et al. (3), Bank et al. (4)).
The directory of T-cell receptors must be able to address the immense diversity of antigenic determinants. This is obtained by genetic recombination of different discontinuous segments of genes which code for the different structural regions of T-cell receptors. Thus, the genes contain V segments (variable segments), optionally D segments (diversity segments), J segments (junction segments) and C segments (constant segments). During the differentiation of T-cells, specific genes are created by recombination of V, D and J segments for the xcex2 and xcex4 loci and V and J segments for the xcex1 and xcex3 loci. These specific combinations as well as the pairing of two chains create the combinational diversity. This diversity is highly amplified by two supplementary mechanisms, namely the imprecise recombination of V-D-J or V-J segments and the addition of nucleotides corresponding to the N region (Davis et al. (5).
A certain number of genetic V segments are already known. These segments have been grouped into subfamilies as a function of the similarity of sequences. By definition, the segments which have more than 75% similarity in the nucleotide sequence have been considered as members of the same subfamily (Crews et al. (6)). The known Vxcex1 nongenetic segments have also been classified into 22 subfamilies, 14 of which have only one member (see Concannon et al. (7), Kimura et al. (8), Wilson et al. (9)).
Moreover, about 60 J genetic segments have been described (9).
Furthermore, monoclonal antibodies directed against specific segments of the variable parts of T-cell receptors, in particular the xcex2 or xcex4 chains, were recently described in WO 90/06758. These monoclonal antibodies are useful not only as diagnostic tools but also as therapeutic tools, for example, vis-à-vis rheumatoid athritis.
The use of synthetic peptides corresponding to the variable regions of the xcex1 or xcex2 chains in the treatment of auto-immune diseases is also described (23 and 24).
It is also known that variations exist from one individual to another in the expression of different variable segments of the T-cell receptor in man (27 and 28).
The present inventions aims to enrich the directory of genetic segments coding for the variable regions of the chains of T-cell receptors by providing on the one hand new Vxcex1 genetic segments belonging to new subfamilies or belonging to subfamilies of which at least one member is already known, and on the other hand, new Jxcex1 genetic segments.
Therefore a subject of the present invention is nucleotide sequences coding for the variable regions of xcex1 chains of human T lymphocyte receptors, corresponding to cDNA""s containing nucleotide sequences chosen from any one of the following:
axe2x80x94Vxcex1 segments corresponding to one of the sequences SEQ ID No. 1 to 11, and
bxe2x80x94Jxcex1 segments corresponding to one of the sequences SEQ ID No. 12, 13 and 15 to 20, and the sequences which differ from them by one or more nucleotides.
More particularly a subject of the present invention is:
sequences coding for the variable regions of xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs containing nucleotide sequences chosen from any one of the Vxcex1 segments corresponding to one of the sequences SEQ ID No. 1 to 10 and the sequences which differ from them by one or more nucleotides,
sequences coding for the variable regions of xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs containing nucleotide sequences chosen from any one of the Jxcex1 segments corresponding to one of the sequences SEQ ID No. 12, 13 and 15 to 20 and the sequences which differ from them by one or more nucleotides.
The expression xe2x80x9cand sequences which differ from them by one or more nucleotidesxe2x80x9d, encompasses alleles which differ by up to 8 nucleotides, but more often differ by 1 or 2 nucleotides or which can differ by the deletion or addition of one or two codons.
Also a more particular subject of the invention is: nucleotide sequences coding for the variable regions of xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs corresponding to all or part of the nucleotide sequences chosen from any one of the Vxcex1 segments corresponding to one of the sequences SEQ ID No. 2 to 5, and the sequences which differ from them by one or two nucleotides,
nucleotide sequences coding for the variable regions of the xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs corresponding to all or part of the nucleotide sequences chosen from any one of the Vxcex1 segments corresponding to one of the sequences
1 to 200 of SEQ ID No. 1
1 to 467 of SEQ ID No. 6
1 to 77 of SEQ ID No. 7
1 to 151 of SEQ ID No. 8
291 to 386 of SEQ ID No. 9
1 to 260 of SEQ ID No. 10
and the sequences which differ from them by one or two nucleotides,
nucleotide sequences coding for the variable regions of the xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs corresponding to all or part of the nucleotide sequence corresponding to SEQ ID No. 11 and which contain the 108 nucleotide,
nucleotide sequences coding for the variable regions of the xcex1 chains of human T lymphocyte receptors, corresponding to cDNAs corresponding to all or part of the nucleotide sequences chosen from any one of the Jxcex1 segments corresponding to one of the sequences SEQ ID No. 12, 13 and 15 to 20 and the sequences which differ from them by one or two nucleotides.
By the expression xe2x80x9cnucleotide sequences corresponding to cDNAs corresponding to all or part of the nucleotide sequencesxe2x80x9d is also designated the complete sequences as well as fragments of these sequences including short fragments (oligonucleotides) which can be used as probes (generally containing at least 10 nucleotides) or as primers (generally containing at least 15 nucleotides). In a general fashion, the invention encompasses the group of new oligonucleotides which are fragments of Vxcex1 and Jxcex1 sequences according to the invention.
As to the sequences which differ by one or two nucleotides, they correspond to variations which are observed experimentally at the time of determination of the nucleotide sequence of several cDNAs.
Also a subject of the present invention is the peptides coded by the nucleotide sequences according to the invention as well as the alleles and the derivatives of the latter which have the same function.
Also a subject of the present invention is the peptides constituted by or composed of a peptide sequence coded by all or part of the sequence 108 to 364 of SEQ ID No. 11.
In a general fashion, the present invention encompasses the peptides constituted by or composed of a peptide sequence coded by the nucleotide sequences according to the invention as well as fragments of these peptides. It also encompasses the peptides which differ from the latter by one or more amino acids and which have the same function. These peptides can correspond to modifications such as those known with muteins or to allelic variations. In fact it has been shown in particular that certain genetic segments coding for the variable regions of chains of T receptors in man were subjected to a phenomenon of genetic polymorphism called allelic variation (25). The present invention encompasses the peptides resulting from this phenomenon.
The nucleotide sequences according to the invention have been obtained according to the following stages:
isolation of the RNA""s of peripheral lymphocytes of an individual;
obtaining the complementary DNA using reverse transcriptase and a primer A which is specific to the Cxcex1 region (SEQ ID No. 21);
genetic amplification (by Anchored Polymerase Chain Reaction or A-PCR) using a DNA polymerase, a poly C primer (SEQ ID No. 22) and a primer B which is specific to the Cxcex1 region (SEQ ID No. 23);
a new amplification by A-PCR using DNA polymerase and a primer C which is specific to the Cxcex1 region (SEQ ID No. 24);
insertion in a plasmid vector;
transformation of a bacterial host with the recombinant vector;
screening of recombinant bacterial colonies with a labelled oligonucleotide D which is specific to Cxcex1 (SEQ ID No. 25);
extraction of plasmids from positive colonies;
and sequencing of DNA fragments containing the Cxcex1 region.
The present invention can be reproduced, in particular, by bispecific genetic amplification (polymerase chain reaction or PCR) by starting with the peripheral lymphocytes which express the mRNA including the variable or junctional segments corresponding to sequences ID No. 1 to 13 and 15 to 20 of the invention or alternatively by applying this PCR technique to genomic DNA of any somatic cell of an individual taken at random. The invention can also be reproduced by preparing the above genetic sequences by the chemical synthesis of oligonucleotides.
The peptides according to the invention can be obtained by standard peptide synthesis. They can also be obtained by the application of known genetic engineering techniques including the insertion of a DNA sequence coding for a peptide according to the invention into an expression vector such as a plasmid and the transformation of cells with this expression vector.
Therefore a subject of the present invention is also plasmids and expression vectors containing a DNA sequence coding for a peptide according to the invention as well as the hosts transformed with this vector.
Also a subject of the present invention is antibodies, and, in particular, monoclonal antibodies directed, against an antigenic determinant belonging to or composed of a peptide according to the invention.
The monoclonal antibodies may be obtained by any of the techniques which allow the production of antibody molecules from cell line culture. These techniques include different techniques using hybridomas.
The antibody production may be obtained in animals by the immunization of the animals by injection with the peptides or fragments according to the invention, whether they be natural, recombinant or synthetic, optionally after coupling to an immunogen such as tetanic anatoxin, or also by injection of human T lymphocytes expressing the corresponding sequences at their surface, including recombinant cells transfected with the corresponding coding sequences.
Also a subject of the present invention is hybridomas producing monoclonal antibodies directed against the polypeptides according to the invention.
The present invention also encompasses the fragments and the derivatives of monoclonal antibodies according to the invention which are reactive with defined variable regions of T-cell receptors. These fragments are, in particular, the F(abxe2x80x2)2 fragments which can be obtained by the enzymatic leavage of antibody molecules with pepsin, the Fabxe2x80x2 fragments which can be obtained by reduction of the disulphide bridges of F(abxe2x80x2)2 fragments and the Fab fragments which can be obtained by the enzymatic cleavage of antibody molecules with papain in the presence of a reducing agent. These fragments can also be obtained by genetic engineering.
The monoclonal antibody derivatives are for example antibodies or fragments of these antibodies to which labellers such as a radio-isotope are attached. The monoclonal antibody derivatives are also antibodies or fragments of these antibodies to which therapeutically active molecules are attached, in particular, cytotoxic compounds.
The products of the invention have several uses in the field of diagnostics and in the field of therapeutics.
1xe2x80x94Uses in the Field of Diagnostics
The oligonucleotides contained in the nucleotide sequences according to the invention can be used to constitute detection probes (generally at least 10. nucleotides) which are capable of hybridizing with a variable region of the xcex1 chain or primers for the amplification of DNA (generally containing at least 15 nucleotides and preferably at least 17 nucleotides) which are capable of being linked to a sequence to be amplified.
Thus the oligonucleotides are used in the diagnosis of immune disorders by detecting the presence of nucleic acid sequences which are homologues of a gene coding for the variable regions of chains of T-cell receptors in the mRNA of a sample from a patient. Different methods can be used to establish a connection between the expression of T-cell genes and an illness. These methods include:
axe2x80x94the production and analysis of cDNA expression libraries-obtained from T-cells connected with the illness to determine the frequency of dominant genes;
bxe2x80x94Southern blot analysis of samples of genomic DNA to determine whether genetic polymorphisms or rearrangements of the genes coding for the T-cell receptors exist;
cxe2x80x94the analysis of samples by obtaining cDNA, amplification by PCR and hybridization with labelled probes;
dxe2x80x94the hybridization in situ of T-cells without culture of T-cells beforehand.
The primers are used in PCR reactions in a method such as that defined in c above.
The monoclonal antibodies, the fragments or the derivatives of these antibodies according to the invention, in particular the anti Vxcex1 antibodies, can be used to study T-type immune responses, for example in the field of the auto-immune diseases of oncology of allergies, of transplants and of infectious diseases. In particular, the directory of different variable xcex1 segments of the T receptor can be studied, whether it be blood or tissue T-cells. In a general fashion the techniques used can be in vitro or in vivo methods.
With in vitro methods, the samples used can be samples of body fluids or tissue samples. The techniques used can include in particular flow cytofluorimetry to analyse blood T lymphocytes or labelling with immunoperoxidase on an anatomopathological section to study the lymphocytes infiltrating the tissues.
With in vivo methods, the antibodies, their fragments or their derivatives are administered by the usual routes, for example by intravenous route, and the immunospecific linkages are detected. This can be obtained for example in the case where an antibody is used which is labelled with a radio-isotope.
2xe2x80x94Uses in the Therapeutic Field
The oligonucleotides contained in the nucleotide sequences according to the invention can be used in therapeutics as anti sense oligonucleotides. In fact it is known that it is possible in vitro to inhibit the expression of a transcript gene in human lymphocytes by incubating these lymphocytes with an anti sense oligonucleotide specific to the gene in question (26). These anti sense oligonucleotides generally contain at least 10 and, preferably, at least 16 nucleotides. These anti sense oligonucleotides can be in particular the inverted and complemented sequences corresponding to 20 nucleotides upstream from the initiation site of the translation (ATG). The significance of the use in vitro of anti sense oligonucleotides specific to a Vxcex1 or Jxcex1 genetic segment is to abolish (or strongly diminish) the expression of a T receptor containing this Vxcex1 or Jxcex1 segment and thus to obtain a phenomenon of clonal deletion at the level of the specific reactivity of T lymphocytes. The anti sense oligonucleotides can not only be used in vitro on human T lymphocytes which are then reinjected, but also in vivo by local or systemic injection preferably after modification to increase the stability in vivo and the penetration into the T lymphocytes of these oligonucleotides.
The monoclonal antibodies according to the invention, in particular the anti Vxcex1 antibodies can be used to modulate the immune system. It is in this way that the antibodies can be administered to block the interaction of the effector T-cells with their specific antigen. Anti T receptor antibodies linked for example to a cytotoxic molecule or a radio-isotope can also be administered in a way so as to obtain a clonal deletion, thanks to the specific fixation on an xcex1 chain of a T-cell receptor. The monoclonal antibodies according to the invention can be used in therapeutics at low mitogenic concentrations so as to activate, in a specific fashion, certain sub-assemblies of T-cells or can be used at much higher concentrations to fix them to the receptors concerned and thus label these sub-assemblies with a view to their elimination by the reticulo-endothelial system. An important criterion in the treatment of an illness is the ability to modulate the sub-assemblies of T-cells linked with an illness. The exact nature of this therapeutic modulation, namely blocking or suppressing a particular sub-assembly of T-cells or on the contrary stimulating and activating a particular sub-assembly, will depend on the illness in question and the specific sub-assembly of T-cells concerned.
This type of treatment has an advantage over current treatments using antibodies such as the treatment with anti CD3 antibodies in patients having had a kidney transplant and having a rejection problem, given that thanks to the invention there will be no modulation of the totality of the T-cell population but only of the sub-assembly of T-cells expressing the xcex1 sub-family specific to the T-cell receptors.
Moreover, as the response of T-cells is often oligoclonal, it is generally convenient to use xe2x80x9ccocktailsxe2x80x9d of several antibodies in therapeutics.
In addition anti Vxcex1 antibodies can be used to select T lymphocytes in vitro, for example by passing through a column containing spheres carrying the antibody. This separation of certain T lymphocytes can be used with a view to culturing these lymphocytes before reinjection into the patient.
Moreover, all or part of the peptide sequences according to the invention can be used in therapeutics, that is to say the peptide sequences coded by the nucleotide sequences according to the invention or fragments of these sequences (generally containing at least 8 to 10 amino acids). These sequences or these fragments, administered to humans or animals, can act as a decoy, that is to say they fix themselves on the epitope carried by the harmful antigen and stop the reaction of normal T-cells with the antigen, preventing in this way the development of an illness which is aggressive towards the self determinants. They can also be used as immunogens in the manufacture of vaccines (optionally after conjugation with protein carriers).